While the normal immune system is closely regulated, aberrations in immune response are not uncommon. A wide variety of medical treatments thus require regulation of the immune response in a patient. For example, T cell-mediated inflammatory diseases are known, in which an inappropriate T cell response is a component of the disease. These include both diseases mediated directly by T cells, and diseases in which an inappropriate T cell response contributes to the production of abnormal antibodies. In some instances, the immune system functions inappropriately and reacts to a component of the host as if it were, in fact, foreign. Such a response results in an autoimmune disease, in which the host's immune system attacks the host's own tissue. T cells, as the primary regulators of the immune system, directly or indirectly affect such autoimmune pathologies.
Heat Shock Proteins and Immunity
During their migration into inflammatory sites, T cells interact with tissue components and encounter a variety of immuno-modulators, including cytokines, chemokines, acute phase proteins, and heat shock proteins (HSP). In addition to serving as a chaperone inside the cell, HSP60 is expressed by cells exposed to stress (Wallin et al., 2002, Kol et al., 1999) or immune activation (van Eden et al., 1988), and is present in the blood and tissues during inflammation (Yokota et al., 2000; Xu et al., 2000; Laplante et al., 1998; Ohashi et al., 2000, Hu et al., 1998; Mor et al., 1992). HSP60 is also involved as an autoantigen in type 1 diabetes and arthritis, and appeal's to down-regulate inflammation in models of these autoimmune diseases (Elias et al., 1997, Quintana et al., 2002, van Eden et al., 1988).
Numerous disclosures claim uses of heat shock proteins or fragments thereof as immune modulators in diagnosis, treatment or prevention of autoimmune diseases. Many of these disclosures relate to the human HSP60 or to its bacterial equivalent HSP65, or fragments thereof.
For example, the particular protein produced by the human body during development of Insulin Dependent Diabetes Mellitus (IDDM, type 1 diabetes), which serves as a diagnostic marker for the incipient outbreak of IDDM, is the human heat shock protein having a size of about 62 kD (human HSP60) or an antigen cross reactive therewith as disclosed in EP 0417271, and in U.S. Pat. Nos. 5,114,844, 5,578,303, 5,671,848, and 5,780,034. The p277 peptide, being the epitope of the human HSP60 involved in IDDM and corresponding to positions 437-460 of the human HSP60 sequence (SEQ ID NO:1), was first described in Israeli Patent No. 94241 of the present applicant. It has been disclosed that fragments of this HSP60 protein, including p277 and derivatives thereof, may serve as therapeutically useful entities in preventing or alleviating IDDM (U.S. Pat. No. 6,180,103, WO 96/19236, and WO 97/01959).
WO 89/12455 and WO 94/29459 disclose the use of stress proteins and analogs for producing or enhancing an immune response or for inducing immune tolerance, for prophylaxis or therapy of autoimmune diseases and for treating or preventing infections or cancers. A fusion protein is claimed comprising a stress protein fused to a protein against which an immune response is desired.
WO 95/25744 discloses microbial stress protein fragments containing epitopes homologous to related mammalian epitopes—used to treat and prevent inflammatory autoimmune diseases and to prevent transplant rejection. The protective epitopes are located in short peptides comprising sequences of 5-15 amino acids of stress proteins that are highly conserved between microorganisms and animals.
WO 97/11966 and WO 96/10039 disclose polypeptides of up to 21 amino acids derived from microbial heat shock protein, which are useful for prophylaxis or treatment of autoimmune diseases especially arthritis.
WO 96/16083 discloses a peptide 25 amino acids long, derived from the 10 kD heat shock protein (HSP10) of Mycobacterium tuberculosis, which is useful in pharmaceutical products for the treatment of inflammatory pathologies, especially rheumatoid arthritis.
WO 91/02542 discloses the use of antigenic and/or immuno-regulatory material derived from Mycobacterium vaccae and specifically HSP60, for treating chronic inflammatory disorders caused or accompanied by an abnormally high release of IL-6 and/or TNF-α.
WO 96/18646 discloses peptides of 9-20 amino acids derived from Mycobacterial HSP60 used for treatment or prevention of autoimmune CNS diseases, e.g. multiple sclerosis, chronic inflammatory CNS disease and primary brain tumors.
WO 94/02509 discloses peptides of 7-30 amino acids derived from DR3-restricted epitope of Mycobacterial HSP60 used for treatment of HLA-DR3 related autoimmune diseases.
U.S. Pat. No. 5,958,416 describes heat shock protein peptides and methods for modulating autoimmune central nervous system diseases.
EP 262710 of Cohen et al. discloses the use of HSP65, or fragments thereof for the preparation of compositions for the alleviation, treatment and diagnosis of autoimmune diseases, especially arthritic conditions. EP 322990 of Cohen et al. discloses that a polypeptide having amino acid sequence 172-192 of HSP65 is capable of inducing resistance to autoimmune arthritis and similar autoimmune diseases. WO 92/04049 of Boog et al. discloses peptides derived from Mycobacterium tuberculosis protein HSP65 containing at least 7 amino acid residues that inhibit antigen recognition by T lymphocytes in treatment of arthritis and organ rejection. The use of p277 in the treatment of arthritic conditions has not been disclosed previously.
WO 02/16549 of Cohen et al. relates to DNA vaccines useful for the prevention and treatment of ongoing autoimmune diseases. The compositions and methods of the invention feature the CpG oligonucleotide, preferably in a motif flanked by two 5′ purines and two 3′ pyrimidines. The vaccines optionally further comprise DNA encoding a peptide or a polypeptide selected from the group consisting of HSP60, p277 or p277 valiants. That disclosure is directed to methods and compositions for the ameliorative treatment of ongoing autoimmune disease in general and Insulin Dependent Diabetes Mellitus (IDDM) in particular.
WO 03/096967 of Cohen et al. discloses DNA vaccines encoding HSP60, HSP70 or HSP90 and active fragments thereof for the treatment of autoimmune diseases such as arthritis.
U.S. Pat. No. 5,993,803 discloses that when HSP60, p277, or other peptides and analogs thereof, are administered in a recipient subject before transplantation of an organ or tissue, autoimmunity to HSP60 is down-regulated, resulting in the prevention or suppression of graft rejection of the transplanted organ or tissue.
WO 00/27870 of Naparstek and colleagues discloses a series of related peptides derived from heat shock proteins HSP65 and HSP60, their sequences, antibodies, and use as vaccines for conferring immunity against autoimmune and/or inflammatory disorders such as arthritis. These peptides are intended according to that disclosure to represent the shortest sequence or epitope that is involved in protection of susceptible rat strains against adjuvant induced arthritis. These sequences further disclose what the inventors identify as the common “protective motif”.
WO 01/43691 provides peptides and peptide analogs capable of acting as antagonists of HSP60 characterized in that they have the ability to reduce or prevent the induction of a pro-inflammatory response of cells of the innate immune system by HSP60.
Apart from the disclosures utilizing the role of HSP60 as an autoantigen involved in the progression of various autoimmune diseases for modulating the development of such diseases, other recent disclosures indicate that HSP60, via Toll-like receptor 2 (TLR-2), may directly inhibit T-cell migration in response to CXCL12 (SDF-lα), and the expression of its receptor, CXCR4 (Zanin-Zhorov et al., 2003). WO 03/070761 of Cohen et al provides novel conjugates comprising HSP60 peptides and their uses in treating immune conditions, particularly inflammatory conditions and autoimmune diseases. These conjugates comprise as a first part an HSP60 epitope that is capable of reacting via TLR2 on T cells and as a second segment a specific peptide capable of eliciting a reaction via a T cell receptor (TcR). Conjugates comprising p277 and a suitable TcR epitope were suggested for the therapy of various diseases, depending on the identity of the TcR epitope.
WO 03/063759 discloses peptides and peptide analogs of heat shock proteins capable of interacting directly with dendritic cells, and pharmaceutical compositions comprising dendritic cells exposed to such peptides and analogs, exemplified by a p277 analog, useful for prevention or treatment of inflammatory disorders and autoimmune diseases or malignancies, viral infections and allergy.
WO 04/098489 discloses HSP60 epitopes capable of binding to LPS or to macrophages and pharmaceutical compositions comprising these novel compounds, useful for prevention or treatment of inflammatory and autoimmune diseases and disorders.
WO 2005/048914, published after the priority dates of the present invention, discloses recombinant constructs encoding active fragments of HSP60, which are effective in treating T cell-mediated inflammatory autoimmune diseases by DNA vaccines. The HSP60 fragments of the disclosed invention are identified by their ability to react with T cells isolated from an animal vaccinated with DNA constructs encoding HSP70 to induce Th2/3 T-cell responses.
None of the background art demonstrates, however, that p277 and analogs thereof may be used to down-regulate T cell mediated inflammation irrespective of the involvement of HSP60 as an autoantigen contributing to the development of the pathology, and without being conjugated to or administered with a second TcR antigen.
Hepatitis
Hepatitis is an inflammatory disease that predominantly affects the liver. The disease is characterized by the initial onset of symptoms such as anorexia, nausea, vomiting, fatigue, malaise, arthralgias, myalgias, and headaches, followed by the onset of jaundice. The disease may also be characterized by increased serum levels of liver aminotransferases aspartate aminotransferase (AST) and alanine aminotransferase (ALT); Quantification of these enzymes in serum indicates the extent of liver damage.
Infectious, autoimmune, as well as non-infectious processes such as chemicals, are among the causes of hepatitis. Examples of infectious diseases affecting the liver include, but are not limited to: (i) viral hepatitis, e.g., hepatitis A, B, C, D, E, and G and (ii) parasitic hepatitis, e.g., Schistosoma mansoni, Schistosoma hematobium, and Schistosoma japonicum. Examples of noninfectious diseases affecting the liver include, but are not limited to, autoimmune diseases, such as autoimmune hepatitis and primary biliary cirrhosis. Other forms of noninfectious hepatitis are caused by hepatotoxic agents such as alcohol, drugs and toxins. Regardless of whether the attack on the liver is infectious, autoimmune or noninfectious, the liver responds to injury by recruiting inflammatory cells into the site of attack. T cells, as the primary regulators of the immune system, play an important role in the pathogenesis of a variety of human liver disorders (Heneghan et al., 2002), including autoimmune liver disease, viral hepatitis (Rosen et al., 2002), and alcoholic liver disease (Chedid et al., 1993). Injection of the T cell mitogenic plant lectin concanavalin A (ConA) is a well-established model to study T cell-mediated hepatitis (Tiegs et al., 1992).
Unfortunately, there are few effective treatments for hepatitis. For example, treatment of autoimmune chronic hepatitis is generally limited to immunosuppressive treatment with corticosteroids. While corticosteroid therapy has been shown to extend life, improve biochemical abnormalities and enhance quality of life in many patients, the beneficial effects of corticosteroids are compensated by the often serious complications and side effects associated with the prolonged treatment therewith. For the treatment of hepatitis B and C, the FDA has approved administration of recombinant interferon alpha. However, for adult patients with hepatitis B infections only about 35% responded to such treatment, and in perinatal infectees only about 10% responded to treatment. For hepatitis C infections, despite apparent short-term success utilizing such therapy, the rate of relapse after termination of treatment is high. In addition, a further difficulty with alpha interferon therapy is that the composition frequently has toxic side effects such as thrombocytopenia, leukopenia, bacterial infections, and influenza-like symptoms, which require reduced dosages for sensitive patients. Other agents used to treat chronic hepatitis B or C include the nucleoside analog ribovirin and ursodeoxycholic acid; however, neither has been shown to be very effective. Consequently, the need remains for finding new and effective drugs and methods for treating hepatitis.
None of the background art demonstrates that HSP60 or a fragment thereof may be an effective agent particularly useful for preventing or treating hepatitis.
There exists a long-felt need for an effective means of curing or ameliorating T cell mediated pathologies, including hepatic disorders. Such a treatment should ideally control the inappropriate T cell response, rather than merely reducing the symptoms. The development of new agents capable of selectively inhibiting the deleterious T cell mediated response with minimal side effects is therefore desirable.